Kotiya Deepak, Rana Manjul, Subbarao N, Puri Niti, Tyagi Rakesh K
Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067, India.
School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
Mol Cell Endocrinol. 2016 Jan 15;420:194-207. doi: 10.1016/j.mce.2015.11.001. Epub 2015 Nov 5.
Pregnane & Xenobiotic Receptor (PXR) is one of the 48 members of the nuclear receptor superfamily of ligand-modulated transcription factors. PXR plays an important role in metabolism and elimination of diverse noxious endobiotics and xenobiotics. Like in case of some nuclear receptors its function may also be differentially altered, positively or negatively, by various post-translational modifications. In this context, regulation of PXR function by SUMOylation is the subject of present investigation. Here, we report that human PXR is modified by SUMO-1 resulting in its enhanced transcriptional activity. RT-PCR analysis showed that PXR SUMOylation in presence of rifampicin also enhances the endogenous expression levels of key PXR-regulated genes like CYP3A4, CYP2C9, MDR1 and UGT1A1. In addition, mammalian two-hybrid assay exhibited enhanced interaction between PXR and co-activator SRC-1. EMSA results revealed that SUMOylation has no influence on the DNA binding ability of PXR. In silico analysis suggested that PXR protein contains four putative SUMOylation sites, centered at K108, K129, K160 and K170. In addition to this, we identified the presence of NDSM (Negative charge amino acid Dependent SUMOylation Motif) in PXR. Substitution of all its four putative lysine residues along with NDSM abolished the effect of SUMO-1-mediated transactivation function of PXR. Furthermore, we show that interaction between PXR and E2-conjugation enzyme UBCh9, an important step for implementation of SUMOylation event, was reduced in case of NDSM mutant PXRD115A. Overall, our results suggest that SUMOylation at specific sites on PXR protein are involved in enhancement of transcription function of this receptor.
孕烷与外源性物质受体（PXR）是配体调节转录因子核受体超家族的48个成员之一。PXR在多种有害内源性物质和外源性物质的代谢及清除过程中发挥着重要作用。与某些核受体类似，其功能也可能因各种翻译后修饰而发生正向或负向的差异改变。在此背景下，SUMO化对PXR功能的调节是当前研究的主题。在此，我们报告人类PXR被SUMO-1修饰，导致其转录活性增强。RT-PCR分析表明，在利福平存在的情况下，PXR的SUMO化还增强了关键的PXR调节基因如CYP3A4、CYP2C9、MDR1和UGT1A1的内源性表达水平。此外，哺乳动物双杂交试验显示PXR与共激活因子SRC-1之间的相互作用增强。电泳迁移率变动分析结果表明，SUMO化对PXR的DNA结合能力没有影响。计算机分析表明，PXR蛋白含有四个假定的SUMO化位点，位于K108、K129、K160和K170。除此之外，我们在PXR中鉴定出了负电荷氨基酸依赖性SUMO化基序（NDSM）。将其所有四个假定的赖氨酸残基与NDSM一起替换，消除了SUMO-1介导的PXR反式激活功能的作用。此外，我们表明，在NDSM突变体PXRD115A的情况下，PXR与E2缀合酶UBCh9之间的相互作用（SUMO化事件实施的重要步骤）减少。总体而言，我们的结果表明，PXR蛋白特定位点的SUMO化参与了该受体转录功能的增强。
